Background

Renal cell carcinoma (RCC) is a seventh ranked malignancy with poor prognosis. RCC is lethal at metastatic stage as it does not respond to conventional systemic treatments, and there is an urgent need to find out promising novel biomarkers for effective treatment. The goal of this study was to evaluate the biomarkers that can be potential therapeutic target and predict effective inhibitors to treat the metastatic stage of RCC.

Methods

We conducted transcriptomic profiling to identify differentially expressed genes associated with RCC. Molecular pathway analysis was done to identify the canonical pathways and their role in RCC. Tissue microarrays (TMA) based immunohistochemical stains were used to validate the protein expression of cyclinD1 (CCND1) and were scored semi-quantitatively from 0 to 3+ on the basis of absence or presence of staining intensity in the tumor cell. Statistical analysis determined the association of CCND1 expression with RCC. Molecular docking analyses were performed to check the potential of two natural inhibitors, rutin and curcumin to bind CCND1.

Conclusion

CCND1 was one of the important upregulated gene identified in microarray and validated by TMA. Docking study showed that CCND1 may act as a potential therapeutic target and its inhibition could focus on the migratory, invasive, and metastatic potential of RCC. Further in vivo and in vitro molecular studies are needed to investigate the therapeutic target potential of CCND1 for RCC treatment.

Renal Cell carcinoma (RCC) is a major health problem and accounts for approximately 1.5 percent of all cancer deaths [1, 2]. It accounts for about 3 % of all cancers and 2-3 % per year increase in global incidence [1, 3]. For RCC treatment, surgery is the best option at advance stage, however, one third of patients develop metastases even after surgery [4]. At metastatic stage, prognosis is very poor because RCC patients hardly respond to conventional existing systemic treatments and leads to death [5]. RCC treatment is a big challenge without identification of new drug targets and effective remedies. Although previous studies have reported role of gene alterations, their expression and deregulation of molecular signals to be linked with cancer initiation and progression, there still lack of curative therapy for RCC [6–8]. Therefore, identification of a potential drug target and prediction of suitable ligand is crucial for the patients with RCC.

The cyclin D members (D1, D2 and D3) bind to CDKs and are required for the hematopoietic cells proliferation and survival and perform a rate-limiting antiapoptotic function in vivo [9]. Cyclin D1 (CCND1) overexpression is predominantly correlated with early cancer onset, tumor progression, shorter cancer patient survival and increased metastases [10–12]. Induction of vascular endothelial growth factor (VEGF) production by CCND1 promotes oncogenesis by increasing growth and angiogenesis, while downregulation of death receptor, Fas by CCND1 causes chemotherapeutic and apoptosis resistance [13]. Overexpression of CCND1 has been previously reported in many cancers including lung cancers [14], esophageal squamous cell carcinoma [15], head and neck cancer [16], pancreatic cancer [17], pituitary cancer [18], and breast cancer [19].

CCND1 is a proto-oncogene and a good biomarker for tumor progression, found to be deregulated in several cancers, including RCC. CCND1 along with associated cyclins activates cyclin-dependent kinases (CDKs) - CDK4 and CDK6. G1-S phase transition during cell cycle, requires phosphorylation of retinoblastoma (Rb) by CDK4 and CDK6. Hyperphosphorylation of Rb allows expression of genes involved in DNA replication and cell division [20–23]. The ability of CCND1 to exhibit oncogenic property and to regulate a critical G1-S transition checkpoint by activating CDK4/CDK6, makes it a potential therapeutic target of RCC [24–28].

Alternative or synergistic anticancer therapies using natural compounds and their derivatives (polyphenols, flavonoids, alkaloids, saponins, etc.) have been extensively studied [29]. Rutin is a flavonol glycoside found in many plants, including buckwheat; tobacco; asparagus, green tea etc. and contributes to the antibacterial [30], hepatoprotective [31], neuroprotective [32] and antioxidant [33] properties of the plant. It is structurally very similar to quercitrin and has been used therapeutically to decrease capillary fragility, to protect blood capillaries, and as ingredients of multivitamin nutritional supplements and alternative herbal remedies. It can attach to iron ion, thereby averting its binding to H2O2 and free radical generation. In addition, rutin acts as an angiogenesis inhibitor and can stall the VEGF in vitro; also has potential anticancerous and antiproliferative property [34, 35].

Curcumin commonly known as turmeric is a phytopolylphenol pigment isolated from the plant Curcuma longa, and possesses a variety of pharmacologic properties like anti-inflammatory, antineoplastic, antiproliferative, anticancer, apoptosis inducer, chempreventive [36, 37]. It can inhibit the reactive-oxygen species formation, cyclooxygenases (COX) and other metabolic enzymes involved in inflammation; and can disrupt cell signal transduction via inhibition of protein kinase C. It can interact with myriad of biomolecules by covalent and non-covalent binding. The H-bonding and hydrophobic interactions, arising from the aromatic and tautomeric structures in addition to the flexible linker group owe for the non-covalent interactions [38]. Curcumin reportedly suppress cyclin D1 expression by promoting proteolysis and down-regulating its expression and causes inhibition of CDK4-mediated phosphorylation of retinoblastoma protein [39]. It has been reported that curcumin-treated cells show decreased expression of CCND1, resulting in low cell growth rate. This curcumin-induced CCND1 mRNA down-regulation is perhaps mediated by induction of BTG2 as well as inhibition of nuclear translocation of NF-kappaB [40].

In this study, expression profiling of RCC (CEGMR data) identified 1490 significantly differentially expressed genes and molecular pathway analysis predicted alteration in many important cancer related pathways. However, the major finding of this study was identification and tissue microarray based validation of CCND1 as important over-expressed gene/proteins of RCC. Overexpression of CCND1 can trigger cancer by activating many pathways, including Wnt/β-catenin signaling pathway and has been shown to exhibit oncogenic property, making it a potential therapeutic target. We, therefore, attempted docking study to show the therapeutic potential of anticancerous natural ligands (rutin and curcumin) against the identified potential drug target (CCND1).

Patients and samples

The study was executed on RCC patients from Saudi Arabia and resected tissue samples were collected from collaborating hospitals of Jeddah during the period 2010–2014. For gene expression analysis, fresh surgically resected tumor and normal tissue were collected and stored in RNALater (Invitrogen/Life Technologies, NY, USA) till RNA extraction. All patients included in the present study were Saudi in origin and diagnosed with clear cell or chromophobe renal cell carcinoma without any prior chemotherapy or radiotherapy exposure.

Ethical approval

Local ethical committee has approved this study (08-CEGMR-02-ETH). Patients were included in the present study only after their prior consent.

Molecular docking was performed using Molecular Docking Server on [42]. The MMFF94 force field geometry optimization method was used for energy minimization of ligand molecule: rutin and curcumin using DockingServer. Gasteiger partial charges were added to the ligand atoms at pH 7.0. Non-polar hydrogen atoms were merged, and rotatable bonds were defined. Rest methodology was followed in sequential manner as previously described [2, 6, 43].

Supporting data availability

Data series (Accession No. GSE781, GSE7023, GSE6344) used in present study are available at NCBI’s Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo/).

This study focused on utilizing transcriptomic profiling to identify biomarkers associated with RCC and conducting molecular docking analysis to assess the interactions between potential target and drugs. We identified CCND1 as important overexpressed gene/proteins of RCC and demonstrated its potential as possible anticancer drug target.

Scatter plot of PCA show grouping of similar type based on genome-wide expression values, as represented as eclipse, where each ball represents one sample. Blue and red is representing RCC and normal kidney tissue

Docking studies

We made a structural attempt to study possible binding of two natural famed ligands with the potential therapeutic drug target, Cyclin D1 for cancer therapeutics. CCND1 protein has a classical double cyclin box domain fold, comprising of 11 alpha-helices [44].

Molecular docking studies predicted good interactions between three dimensional structure of drug target (CCND1, PDBID: 2w96) and selected ligands; rutin and curcumin. Molecular docking revealed that both the compounds are able to bind in the ligand binding domain. In silico docking studies revealed interaction of two active compounds with the common vital ligand binding site residues (Leu91, Lys149, Asn151) of cyclin D1. Both rutin and curcumin docked at a common ligand binding site of CCND1 slightly varied intensity as estimated by their size, structure, stereochemistry (Figs. 7 and 8; Table 4). We also examined their complete interaction profile including hydrogen bonds, HB plot, polar, hydrophobic, pi-pi and cation-pi interactions. The estimated free energy of binding with Cyclin D1 for rutin was −4.26 kcal/mol and for curcumin was −4.67 kcal/mol which is very similar, however, the estimated inhibition constant (Ki) was 757.57 μM and 380.02 μM respectively.

Fig. 7

Molecular docking conformation and interactions of rutin and curcumin with Cyclin D1

RCC is a complex heterogeneous tumors involving altered genes and proteins. We performed a transcriptional profiling and functional analysis of RCC to understand the role of identified significant genes in regulation of physiological processes through biological pathways/networks. We found CCND1 as one of the significantly expressed gene and potential biomarker RCC.

HEY1, an upregulated gene, has been reported to be mediator of notch signaling, showing pro-oncogenic function and promotes cancer progression [45, 46]. Neuropilin-2 (Nrp2) is a well known receptor for the vascular endothelial growth factor-C (VEGF-C) and activates lymph nodes as well as promotes tumor metastasis by lymphangiogenesis [47, 48]. LEF1 interacts with β-catenin and plays critical role in proliferation of RCC by activating downstream target genes [49, 50]. Wnt/β-catenin signaling, found activated, regulates embryonic development and is involved in many diseases including cancer, polycystic kidney disease [51–54]. WNT signal and its paracrine mode to growth of cancer cells makes it clinically important to understand the metastasis of tumor cells [53, 55, 56]. HIST1H3H is frequently altered chromatin factors in many cancers [57, 58]. Aldolase, a family member of glycolysis enzymes, was found to be significantly affecting RCC. Aldolase-A was reportedly upregulated while aldolase-B was downregulated in RCC and human primary hepatocellular carcinoma [59–62]. SLC12 family members are involved in regulation of cell volume, blood pressure and chloride concentration, and play a critical role in diseases like cancer, epilepsy and osteoporosis [63]. In the present study, SLC12 was down regulated that is in accordance to other findings [64]. CALB1 is reported to be altered in RCC and found to be negatively stained compared to normal tissue [61, 65].

CCND1 was overexpressed in our as well as other transcriptomics studies [66–69]. We validated CCND1 overexpression by using tissue microarray platform and in silico docking analysis was done to check its therapeutic potential as it plays a key role in G1-S phase transition of cell cycle. There are reports of anti-proliferative, apoptosis inducing and chemopreventive effects of natural bioactive flavonoids like baicalein, catechin, genistein, quercetin, and rutin. Docking analysis showed that rutin and curcumin binds to CCND1 and can potentially inhibit downstream CCND1/CDK4/CDK6 complex formation, required for G1-S phase transition. Our finding demonstrate the anticancer drug targets potential of CCND1 and rutin and curcumin as potential inhibitors, however, this in silico docking study has to be validated further.

Our microarray and immunohistochemistry results suggest significantly high levels of cyclin D1 expression in RCC. Distinct transcriptomic signatures identified for RCC needs verification at larger dataset and additional significant genes need to be further validated for identification of novel biomarkers. The critical role of CCND1 in RCC metastasis by activating G1-S transition of cell cycle has drawn our attention to examine its potential as anticancer drug target. Our in silico docking study shown CCND1 protein as an attractive anticancer target and natural flavanoids rutin and curcumin as potential anticancer drug of RCC and they may be promising in the prevention of kidney cancer too. Quantitative structure-activity relationship studies, ligand binding, efficacy and toxicity should be further investigated before clinical trials. Clinical and therapeutic applications of these natural ligands were initially limited by their low solubility and bioavailability but combination with adjuvant and nano-technology based delivery vehicles can immensely improve their potential. Moreover, these are reported to act in synergism with several other natural compounds or synthetic agents routinely used in chemotherapy and can assist in cancer prevention and treatment when used alone or in combination with other conventional treatments.

Acknowledgment

This project and publication was supported by the NSTIP strategic technologies program in the Kingdom of Saudi Arabia – Project No (10-BIO1258-03, 09-BIO1073-03, 08-MED120-03). The authors also acknowledge with thanks Science and Technology Unit, King Abdulaziz University for technical support. Authors would like to acknowledge the Deanship of Scientific Studies, King Abdulaziz University, Jeddah, Saudi Arabia for funding the research (HiCi-1434-117-2). We also thank the patients, physicians, nurses, and pathologists of the King Abdulaziz University Hospital, and King Faisal Specialist Hospital and Research Center, Jeddah, Saudi Arabia.

Availability of data and material

Authors’ contributions

SK, JM and HF participated in the study design. HJS, AAA, FA and MS performed data collection, DNA extraction and microarray studies. AB and JM did tissue microarray, immunohistochemistry and pathological studies. SK, ZM and HN analyzed data, interpreted the results and drafted the manuscript. KA, AMA, AC, AS and MHQ participated in critical review, editing and finalization of manuscript. All authors have read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

Ethical approval

Local ethical committee has approved this study (08-CEGMR-02-ETH). Patients were included in the present study only after their prior consent.

Ethical approval information is included in the materials and methods section. Local ethical committee has approved this study under the approval number 08-CEGMR-02-ETH.

Consent for publication

Patients’ consent information are included in the material and methods section. Patients were only included in the present study after their prior consent.

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